Project Summary/Abstract The Lamin B receptor (LBR) is a polytopic membrane protein residing at the nuclear envelope in close association with the nuclear lamina. We discovered that human LBR has an essential role in cholesterol synthesis, which is strongly perturbed by LBR mutations responsible for the congenital disorders Greenberg Dysplasia and Pelger-Hut anomaly. Several of these disease-associated mutations lead to the rapid degradation of LBR at the inner nuclear membrane (INM), a site that is poorly understood from the perspective of protein quality control. Progress in this area has been hampered by the absence of suitable methodologies, with the lack of model substrates being the major limitation. This deficiency in our understanding of cellular quality control presents a major obstacle towards understanding and treating congenital diseases caused by mutations in nuclear envelope-resident proteins, commonly referred to as nuclear envelopathies. A detailed characterization of the protein turnover mechanisms responsible for the elimination of LBR disease mutants has allowed us to directly observe inner nuclear membrane protein turnover (INMPT) in mammalian cells for the first time. We have since established LBR-based model substrates and readouts, allowing us to initiate an unprecedented interrogation of protein turnover mechanisms in the nuclear envelope. As a logical extension of these endeavors, we now propose to exploit our newly established experimental platform to identify the machinery responsible for protein quality control at the INM. Two independent yet synergistic approaches will be pursued to achieve this goal: (1) Based on our established strategies to intersect the degradation of LBR derivatives at distinct stages of membrane extraction and proteasomal degradation, we will isolate stalled degradation intermediates to identify associated factors involved in INMPT via mass spectrometry; (2) We will conduct an unbiased, genome-wide CRISPR/Cas9-based screen to discover genes implicated in INMPT. To this end, we will utilize a cell line expressing a degradation-prone, split-GFP derivative of LBR. Mutant stabilization upon functional inactivation of a gene required for INMPT will lead to an increase in fluorescence, allowing us to identify the targeted gene via FACS analysis and next- generation sequencing. For candidates arising from both approaches (1) and (2), we will (3) functionally characterize these factors using our firmly established experimental platform including (i) readouts for localization to the nuclear envelope; (ii) assays for membrane dislocation/extraction and (iii) ubiquitination and proteasomal degradation, thus allowing us to assign a function to each validated candidate gene or interacting protein. The results of these studies will allow us to define the protein homeostasis network that safeguards protein integrity at the nuclear envelope. Apart from closing a major gap in our understanding of cellular protein quality control?an area of significant biomedical relevance?our findings are expected to have important ramifications for our understanding and the treatment of nuclear envelopathies.